IDENTIFICATION OF AN SNRNP-ASSOCIATED KINASE-ACTIVITY THAT PHOSPHORYLATES ARGININE SERINE RICH DOMAINS TYPICAL OF SPLICING FACTORS

The U1 snRNP-specific 70K protein is one of the few snRNP proteins fro m higher eukaryotic cells that is phosphorylated in vivo (1,2). Immuno affinity purified spliceosomal snRNPs (U1, U2, U5, and U4/U6) were tes ted for their ability to phosphorylate in vitro the U1-specific 70K pr otein. An snRNP-associated kinase activity which phosphorylates all U1 -70K isoelectric variants was identified. Like its in vivo counterpart , this snRNP-associated enzyme phosphorylates solely serine residues o f the 70K protein, preferentially utilizing ATP as a phosphodonor. Try ptic phosphopeptide analysis revealed an overlapping set of at least f our radiolabeled peptides in the in vivo and in vitro phosphorylated p rotein, suggesting that the snRNP-associated serine kinase is responsi ble, at least in part, for the 70K protein phosphorylation observed in vivo. Chymotryptic digestion of in vitro, P-32-labeled 70K protein an d in vitro phosphorylation studies with a synthetic peptide, indicated that the multiple 70K phosphorylation sites are limited to a highly c harged, C-terminal domain of the protein. In vitro phosphorylation stu dies with the splicing factor ASF/SF2 and several deletion mutants dem onstrated that, similar to the U1-70K protein, the snRNP-associated se rine kinase phosphorylates the carboxy terminal RS-rich domain of ASF/ SF2. A potential general role for this enzyme in the phosphorylation o f splicing factors and its consequences for pre-mRNA splicing regulati on are discussed.